ABSTRACT: PROJECT 1 This project will develop and evaluate a comprehensive and integrated suite of analytical, computational, and bioassay based approaches for assessing overall reductions in toxicity resulting from bioremediation of Superfund (SF) sites. These tools will then be applied to optimize biodegradation of two contaminant mixtures, triazine herbicides and polycyclic aromatic hydrocarbons representative of environmental exposures faced by our community partners the Yurok Tribe, through systematic investigation of carbon sources, electron acceptors, and reactor detention times. Although both of these contaminant mixtures are known to biodegrade, transformation products (TPs) accumulate and are widely found in groundwater (triazines) and/or have increased toxicity compared to parent compounds (PAHs). Bioreactor performance will be characterized by measuring shifts in microbial community composition, bioassay activity, and both target and nontarget chemical concentrations measured with GC and LC high resolution mass spectrometry (HRMS). This combination of measurements will provide unique insights into interactions among contaminant transformations, microbial populations and overall reductions in human and ecosystem risks. Novel enzyme engineering approaches will be used to identify rate limiting steps in triazine mineralization and to isolate or design improved enzymes to carry out these steps. Microorganisms with improved ability to degrade triazines will be prepared and tested in the bioreactors to assess ability to remove target compounds and to reduce overall bioactivity compared to standard enrichment approaches. Our central hypothesis is that chemical hazard reduction during SF site remediation can be best characterized through broad consideration of both contaminant destruction and byproduct formation. We further hypothesize that a minimum suite of high- throughput assays can be defined to effectively capture the overall risk reduction during remediation and that this suite of assays can guide optimization of bioreactor design and operation. This project will support a paradigm shift in the SRP away from reducing concentrations of specific constituents and toward the overall reduction of deleterious biological effects. The project is strongly integrated with the overall program, drawing on HRMS, metabolomics, and statistical expertise in the Analytical Core, the full range of bioassays available in the Bioanalytical Core, immunoassays from Project 3 especially for triazines and TPs, as well as integrative bioassays for ER and oxidative stress being developed by Projects 4 and 5. The bioassay suite developed here will be used to analyze environmental samples collected through the Community Engagement Core and the overall workflow will be transferred to a broader user community with the assistance of the Research Translation Core.